ABSTRACT- Background: Apaziquone (EO9) is a novel analogue of mitomycin C. EO9 is bioreduced by intracellular reductases into active DNA damaging moieties and may selectively sensitize hypoxic tumor cells. Past clinical trials indicated that systemically administered EO9 was poorly delivered to tumor, while EO9 delivered locally showed significant anti-tumor activity in various xenograft models. Ongoing clinical trials investigating EO9 in superficial bladder tumors with local drug delivery show promising response rates. To date, EO9 has only been used as a single agent. The purpose of this study was to explore EO9 as a radiation sensitizer. Methods: U-87 human glioblastoma cells were injected subcutaneously into the hind limb of athymic NCR NUDE mice and allowed to grow to a hypoxic volume of 500 mm3 before treatment. EO9 (3 days X 2 mg/kg) or vehicle (DMSO) was administered 30 min after each radiation fraction (3 days x 7.5 Gy) on day 1, 2, and 3. Tumor growth analysis was performed by using mixed-effects linear regression to model the base-10 logarithm of tumor volume as a function of time. Results: Tumor growth rate in the vehicle treated group corresponded to a doubling time of 3.2 days. EO9 alone or radiation alone increased tumor doubling time by 1.4 days (p<0.001 vs. control) or 5.2 days (p<0.001 vs. control), respectively. Combination of EO9 and radiotherapy increased the mean doubling time by 8.5 days to 11.7 days, a stronger effect than that seen by a comparable regimen of EO9 alone (p<0.001), or radiation alone (p=0.027 comparing days 1-7). Western blot analysis showed cytochrome P450 reductase was elevated in hypoxic tumors and the enzyme expression was increased by fractionated radiation. Bioreduction of EO9 by the single electron reductase, cytochrome P450 reductase, should target hypoxic tumor cells. Conclusions: These results indicate for the first time that EO9 can benefit a fractionated regimen of radiotherapy and should be explored clinically as a radiation sensitizer. Furthermore, hypoxia and radiation can alter the bioreductive enzyme profile and favor hypoxic cell kill by EO9. Future studies will address optimal scheduling of EO9 with radiation as well as systemic delivery of EO9 in combination with radiation therapy. (This work was supported in part by an unrestricted grant from Spectrum Pharmaceuticals)